Method of producing recombinant TAT-HOXB4H protein for use as a stimulant of hematopoiesis in vivo

ABSTRACT

The present invention relates to a new and nonobvious method of producing the C-terminal histidine tagged TAT-HOXB4 fusion protein (TAT-HOXB4H), providing unexpected benefits of increased yield and stability to allow for in vivo administration of this protein, and pharmaceutical composition comprising an effective ingredient, TAT-HOXB4H, having stimulatory activity on the production of hematopoietic cells. More specifically, recombinant TAT-HOXB4H protein enhances engraftment of bone marrow transplants, hematopoietic reconstruction, bone marrow re-population and number of circulating stem cells, particularly after chemotherapy or irradiation.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.12/042,097, filed on Mar. 4, 2008, which is incorporated herein byreference.

FIELD OF THE INVENTION

The present invention relates to a new and nonobvious method ofproducing the C-terminal histidine tagged TAT-HOXB4 fusion protein(TAT-HOXB4H) containing at least 6 histidine (SEQ ID NO: 5) residues atthe C-terminus. The method of production provides unexpected benefits ofincreased stability and yield, which allows for successful in vivoadministration of this protein.

BACKGROUND OF THE INVENTION

The growing interest in regenerative medicine has fueled the search fororgan-specific stem or self-renewing cells. The best studied populationof self-renewing cells is the hematopoietic stem cells (HSCs) as theyare innovative options for treatment of diseases from cancer tometabolic disease to immunodeficiencies.

The process of blood cell formation whereby red and white blood cellsare replaced through the division of HSCs located in the bone marrow iscalled hematopoiesis. The HSCs have the key properties of being able toself-renew and to differentiate into mature cells of both lymphoid andmyeloid lineages. However, the genetic mechanisms responsible for thecontrol of self-renewal and differentiation outcomes of HSC divisionsremain largely unknown.

Currently, transplantation of human HSCs from adult bone marrow,mobilized peripheral blood, and umbilical cord blood (UCB) has been usedclinically to treat hematopoietic cancers (leukemias and lymphomas) andto aid immune system recovery from high-dose chemotherapy ofnon-hematopoietic cancers. However, efficient transplantation requiressubstantial amount of HSCs from different sources and may requireexpansion.

HSCs can be originated from bone marrow, peripheral blood and UCB. Theextraction of bone marrow cells requires surgery and painful procedure,and therefore becomes less favorable approach. Using peripheral bloodcells is also a problem because the difficulty of obtaining qualifiedHSCs from the hematopoiesis compromised patient who suffer from illnessor chemotherapy. UCBs are relatively easier obtained and the quality ofHSCs is much higher, however, the number of HSCs obtained from thisapproach is still limited. Cell number from each extraction issufficient for a child, but may be insufficient for an adult. Toovercome this potential problem, a new approach that facilitates HSCproliferation in vitro by intervening stem cell self-renewal process isindeed necessary for HSC transplantation.

It has been indicated that transcription factors play a critical role inthe regulation of gene expression and the differentiation in stem cells(Orkin, S. H. Nature Reviews Genetics 1, 57-64, 2000). Transcriptionfactor switches various cellular processes through binding to specificgene target, and this regulation also depends on its cellularconcentration. A group of transcription factors called DNA bindinghomeobox (HOX) was previously found to play a major role inembryogenesis. Recently, HOX family is also found to be involved in thedevelopment of HSCs (Buske, C. et. al., J. Hematol. 71, 301-308, 2000).The regulation of HSC self-renewal by HOX transcription factor wasstudied by. Dr. Guy Sauvageau from the University of Montreal.Sauvageau's group showed that homeobox gene HOXB4 is critical in theregulation of HSC self-renewal for its ability of maintaining HSCpopulation in bone marrow. HOX genes expressed in blood cells was firstobserved in human and mouse cell lines. Some types of HOX genes areexpressed ubiquitously in various cell types, while others arespecifically expressed in certain type of cells or certain time pointsduring the development. For example, eight members of human HOXB clusterare expressed in early stage of erythrocyte development. However, HOXBgenes such as HOXB4 and HOXB7 are also expressed in T cells and B cells.Sauvageau's group confirmed that nine HOXA, eight HOXB and four HOXCgenes are expressed in CD34⁺ bone marrow cells. Among these CD34⁺ bonemarrow cells, HOXB2, HOXB9 and HOXA10 are most enriched in erythrocyteprogenitor cells. However, no HOX genes are expressed in CD34⁻ cells.Human homeobox B4 (HOXB4) gene was recently demonstrated to effectivelyexpand HSCs in a retroviral or recombinant protein form. RecombinantTAT-HOXB4 proteins were used to expand stem cells in the laboratoryscale without the risk of retroviral insertion or co-culture with bonemarrow stromal cells (See Krosl, J. et al., Nature Medicine 9,1428-1432, 2003). Therefore, HOXB4 protein is regularly used as astimulant to promote HSCs expansion in vitro (FIG. 1).

Recent evidence indicated that by adding a TAT protein sequence tag atthe N-terminus of HOXB4, exogenous HOXB4 can be delivered into the cell.This TAT sequence directs the transportation of HOXB4 from extracellularside into intracellular side. Upon entering the cytosol, HOXB4 can berefolded into its native conformation by chaperon HSP90. TAT-HOXB4 isable to promote HSC proliferation to 2-6 fold (Amsellem, S. et. al.,Nature Medicine 9, 1423-1427, 2003; Krosl, J. et. al., Nature Medicine9, 1428-1432, 2003). However, the yield of recombinant TAT-HOXB4 proteinfrom E. coli by using regular purification procedure is too low.

In an effort to increase the yield of the recombinant TAT-HOXB4 protein,a method of making a TAT-HOXB4H protein with additional six histidine(SEQ ID NO: 5) residues tagged at the C-terminus was developed whichresulted in 3-4 fold yield compared to that of the original proteinafter purification. The resultant recombinant protein (TAT-HOXB4H)contains 6 histidine (SEQ ID NO: 5) residues at the C-terminus. Thismethod was described in detail in the PCT application PCT/CN2006/000646.

It was shown that the recombinant TAT-HOXB4H protein can be used toexpand human peripheral blood or UCB stem cells and the expanded stemcells still possess their pluripotency. Furthermore, the stem cellstreated with the recombinant TAT-HOXB4H protein incorporated into thebone marrow of nonobese diabetic-severe combined immunodeficiency(NOD-SCID) mice and human leukocytes were detected in peripheral whiteblood cells, indicating immune and hematopoiesis reconstitution in themice.

However, recombinant TAT-HOXB4H proteins have never been used before asa stimulator of hematopoiesis in vivo, specifically, to enhancehematopoietic reconstitution, expansion, bone marrow re-population andto increase the number of peripheral circulating stem cells,particularly after chemotherapy or irradiation. Krosl et al. (2003) andAmsellem et al. (2003) were not able to obtain large amounts of highlystable HOXB4 protein to be used in clinical studies to expand HSCs. Inthe present invention, the total amount of TAT-HOXB4H protein obtainedafter purification generally ranges from 6-10 mg from a 1 liter culture,while the total amount of TAT-HOXB4 protein obtained after purificationfrom a 1 liter culture using prior art methods generally ranges from 1-2mg. The pTAT-HA-HOXB4 plasmid used to express the TAT-HOXB4 proteinusing prior art methods was a gift from Dr. Guy Sauvageau, University ofMontreal, Canada. The method of purifying the TAT-HOXB4H protein usingthe present invention clearly indicates the increased yield of proteinnecessary for the in vivo administration. Krosl et al. (2003) alsoreported that most of their TAT-HOXB4 protein was lost after 4 h ofincubation in medium with serum. The present invention shows asignificantly high stability of TAT-HOXB4H protein even after 4 weeks,which is a key factor for the use of TAT-HOXB4 protein in clinicalstudies.

SUMMARY OF THE INVENTION

The present invention is based on the new and nonobvious method ofproducing the TAT-HOXB4H protein with high yield and stability, and onthe finding that recombinant TAT-HOXB4H protein, when administered to asubject in need thereof, increase number of HSCs in both the bone marrowand peripheral blood in vivo.

One aspect of the invention relates to a method of producing aTAT-HOXB4H protein. The method comprises: (a) providing a host cellcomprising a vector encoding the protein; (b) expressing the protein inthe host cell; (c) collecting an impure solution of the expressedprotein; (d) purifying the protein from the solution by: (i) applyingthe solution to a chromatography column for purifying histidine-taggedproteins sold under the trademark HISTRAP^(TM); (ii) washing thechromatography column for purifying histidine-tagged proteins; (iii)eluting the partially purified protein from the chromatography columnfor purifying histidine-tagged proteins to form a partially purifiedprotein solution; (iv) applying the partially purified protein solutionto a cation ion exchange chromatography column sold under the trademarkMONOSP™; (v) washing the cation ion exchange chromatography column; (vi)eluting the purified protein from the cation ion exchange chromatographycolumn in denatured form; (e) refolding the eluted denatured proteinusing hydrophobic compounds by (i) combining the eluted denaturedprotein and a solution of hydrophobic compounds to form a solution ofprotein and hydrophobic compounds; (ii) desalting the solution ofprotein and hydrophobic compounds to obtain a desalted protein andhydrophobic compound solution; (iii) removing the hydrophobic compoundsfrom the desalted protein solution using ultrafiltration.

One aspect of the invention relates to a method for enhancing themobilization of HSCs from bone marrow to peripheral blood. The methodcomprises: a) administering an effective amount of a TAT-HOXB4H proteinproduced by the methods described herein to a subject in need thereof,and b) allowing the TAT-HOXB4H protein to increase the absolute numberof hematopoietic stem cells in the bone marrow of the subject therebyenhancing the mobilization of hematopoietic stem cells to the peripheralblood of the subject.

Another aspect of the invention relates to a method for improving therecovery time of a patient having undergone HSC transplantation,irradiation or chemotherapy. The method comprises: a) administering aneffective amount of a TAT-HOXB4H protein produced by the methodsdescribed herein to a subject in need thereof, and b) allowing theTAT-HOXB4H protein to increase the absolute number of HSCs to the bonemarrow of the subject.

One aspect of the invention relates to a pharmaceutical composition formobilization of HSCs from bone marrow to peripheral blood in a subjectin need thereof. The pharmaceutical composition of the inventionincludes an effective amount of a TAT-HOXB4H protein produced by themethods described herein sufficient to increase the absolute number ofHSCs in the bone marrow of the subject thereby enhancing themobilization of HSCs to the peripheral blood of the subject.

The pharmaceutical composition of the invention may be administered to apatient having undergone autologous HSC transplantation for improvingthe recovery time after HSC transplantation.

The pharmaceutical composition of the invention may be administered to agranulocyte-colony stimulating factor (G-CSF)-insensitive patient as asubstitute for G-CSF for mobilization of HSCs to peripheral blood.

The pharmaceutical composition of the invention may be administered to aHSC donor thereby allowing a sufficient amount of HSCs to be collectedfor transplantation in a much less invasive procedure from theperipheral blood rather than the bone marrow of said donor.

Another aspect of the invention relates to treatment of diseases causedby inherited HSC deficiency by systemically administering an effectiveamount of a recombinant TAT-HOXB4H protein produced by the methodsdescribed herein or of a pharmkeutical composition comprising the sameto a subject suffering from the diseases. The administered recombinantTAT-HOXB4H protein thereby increases the absolute number of HSCs in thebone marrow of the subject.

A further aspect of the invention relates to a method for improving therecovery time after HSC transplantation by systemically administering aneffective amount of a recombinant TAT-HOXB4H protein produced by themethods described herein or of a pharmaceutical composition comprisingthe same to a subject in need thereof.

A still further aspect of the invention relates to a method forenhancing HSC recovery of a patient receiving irradiation orchemotherapy by systemically administering an effective amount of arecombinant TAT-HOXB4H protein produced by the methods described hereinor of a pharmaceutical composition comprising the same to a subject inneed thereof.

BRIEF DESCRIPTION OF THE FIGURES

The foregoing aspects and advantages of this invention may becomeapparent from the following detailed description with reference to theaccompanying drawings in which:

FIG. 1 shows a schematic representation of mobilization of HSCs fromCD34 cells to peripheral blood (PB) by in vivo expansion.

FIG. 2 shows schematic representation of construction and cloning ofpTAT-HOXB4H in modified pET21b vector. (The six His tags are disclosedas SEQ ID NO: 5)

FIG. 3 represents the DNA sequence (SEQ ID NO: 3) of pTAT-HOXB4H. Theadditional 6 histidine (SEQ ID NO: 5) residues introduced at the N- andC-termini in pTAT-HOXB4H are underlined and TAT is highlighted.

FIG. 4 shows the protein sequence (SEQ ID NO: 4) of TAT-HOXB4H protein.

FIG. 5 shows 10% SDS-polyacrylamide gel (1.5 mm) demonstration of thepurification of TAT-HOXB4H protein. The SDS-polyacrylamide gel wasstained with coomassie blue. Lane 1, molecular weight markers (M), 0.3μg protein; lane 2, cell lysate from uninduced BL21(DE3)pLysS cellsexpressing TAT-HOXB4H protein, 1 μg protein; lane 3, cell lysate frominduced BL21(DE3)pLysS cells expressing TAT-HOXB4H protein with 1 mMIPTG, 1 μg protein; lane 4, purified TAT-HOXB4H, 0.7 mg protein; lane 5,purified TAT-HOXB4 (0.2 mg protein). The pTAT-HA-HOXB4 plasmid used toexpress the TAT-HOXB4 protein (lane 5) was a gift from Dr. GuySauvageau, University of Montreal, Canada. Equal volume of fractions,that were collected froth cation ion exchange chromatography column,were loaded in the lanes 4 and 5.

FIG. 6 shows SDS-polyacrylamide gel analysis of the stability ofpurified TAT-HOXB4H protein stored in PBS for 0 hours (A) and 16 hours(B) at 4° C. M represents molecular weight markers, 0 represents 0 hoursincubation at 4° C., 16 represents 16 hour incubation at 4° C.

FIG. 7 shows the stability of TAT-HOXB4H protein stored at 4° C. and−20° C. in PBS and storage buffer, IMDM, analyzed by 10%SDS-polyacrylamide followed by coomassie staining. Arrows indicateTAT-HOXB4H protein bands.

FIG. 8A shows the stimulatory effect of G-CSF on the number of CD34⁺stem cells in bone marrow of mice analyzed by a flow cytometer.

FIG. 8B shows the effect of PBS on the number of CD34⁺ stem cells inbone marrow of mice analyzed by a flow cytometer.

FIG. 8C shows the stimulatory effect of TAT-HOXB4H on the number ofCD34⁺ stem cells in bone marrow of mice analyzed by a flow cytometer.

FIG. 9A shows the stimulatory effect of G-CSF on the number of CD34⁺stem cells in bone marrow of rhesus monkey analyzed by a flow cytometer.

FIG. 9B shows the stimulatory effect of TAT-HOXB4H together with G-CSFon the number of CD34⁺ stem cells in bone marrow of rhesus monkeyanalyzed by a flow cytometer.

FIG. 9C shows the stimulatory effect of TAT-HOXB4H on the number ofCD34⁺ stem cells in bone marrow of rhesus monkey analyzed by a flowcytometer.

FIG. 9D shows the effect of PBS on the number of CD34⁺ stem cells inbone marrow of rhesus monkey analyzed by a flow cytometer.

FIG. 10 shows effect of TAT-HOXB4H protein on hematopoietic recovery inNOD-SCID mice.

FIG. 11 shows effect of TAT-HOXB4H protein on hematopoietic recovery inBalb/c mice after cisplatin chemotherapy.

DETAILED DESCRIPTION I. The TAT-HOXB4H Protein

The present invention relates to a new and nonobvious method ofproducing the TAT-HOXB4H protein, providing unexpected benefits ofincreased stability and yield, which allows for in vivo administrationof this protein. The TAT-HOXB4H protein is a construct comprising threeelements: TAT, HOXB4, and a histidine tag. HOXB4 is a member of the HOXfamily of transcription factors and promotes HSC expansion. TAT allowsthe HOXB4 moiety to be transported into the cell. The histidine tagallows for initial increased yield from recombinant expression sources,although the method of production further increases the yield of theprotein. pTAT-HOXB4H has been constructed as shown in FIG. 2, and theDNA sequence is shown in FIG. 3. The recombinant TAT-HOXB4H proteinrefers to a TAT-HOXB4 fusion protein with additional six histidine (SEQID NO: 5) residues tagged at the C-terminus (FIG. 4).

Unless otherwise indicated, a protein's amino acid sequence (i.e., its“primary structure” or “primary sequence”) may be written fromamino-terminus to carboxy-terminus. In non-biological systems (forexample, those employing solid state synthesis), the primary structureof a protein (which also includes disulfide (cysteine) bond locations)can be determined by the user.

A “deletion” refers to a change in an amino acid or nucleotide sequencedue to the absence of one or more amino acid residues or nucleotides.The terms “insertion” or “addition” refer to changes in an amino acid ornucleotide sequence resulting in the addition of one or more amino acidresidues or nucleotides, respectively, to a molecule or representationthereof, as compared to a reference sequence, for example, the sequencefound in the naturally occurring molecule. A “substitution” refers tothe replacement of one or more amino acids or nucleotides by differehtamino acids or nucleotides, respectively.

Sequences similar or homologous (e.g., at least about 85% sequenceidentity) to the sequences disclosed herein are also part of thisapplication. In some embodiment, the sequence identity can be about 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher. Alternatively,substantial identity exists when the nucleic acid segments may hybridizeunder selective hybridization conditions (e.g., highly stringenthybridization conditions), to the complement of the strand. The nucleicacids may be present in whole cells, in a cell lysate, or in a partiallypurified or substantially pure form.

Calculations of “homology” or “sequence identity” between two sequences(the terms are used interchangeably herein) are performed as follows.The sequences are aligned for optimal comparison purposes (e.g., gapscan be introduced in one or both of a first and a second amino acid ornucleic acid sequence for optimal alignment and non-homologous sequencescan be disregarded for comparison purposes). In a preferred embodiment,the length of a reference sequence aligned for comparison purposes is atleast 30%, preferably at least 40%, more preferably at least 50%, evenmore preferably at least 60%, and even more preferably at least 70%,80%, 90%, 100% of the length of the reference sequence. The amino acidresidues or nucleotides at corresponding amino acid positions ornucleotide positions are then compared. When a position the firstsequence is occupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position (as used herein amino acid or nucleic acid“identity” is equivalent to amino acid or nucleic acid “homology”). Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences, taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In a preferred embodiment, the percent identity between twoamino acid sequences is determined using the Needleman and Wunsch((1970) J. Mol. Biol. 48:444-453) algorithm which has been incorporatedinto the GAP program in the GCG software package, using either a Blossum62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6,or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet anotherpreferred embodiment, the percent identity between two nucleotidesequences is determined using the GAP program in the GCG softwarepackage, using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60,70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A particularlypreferred set of parameters (and the one that may be used if thepractitioner is uncertain about what parameters may be applied todetermine if a molecule is within a sequence identity, or homologylimitation of the invention) are a Blossum 62 scoring matrix with a gappenalty of 12, a gap extend penalty of 4, and a frame shift gap penaltyof 5. The percent identity between two amino acid or nucleotidesequences can also be determined using the algorithm of E. Meyers and W.Miller ((1988) CABIOS, 4:11-17) which has been incorporated into theALIGN program (version 2:0); using a PAM120 weight residue table, a gaplength penalty of 12 and a gap penalty of 4.

II. Methods of Making the Recombinant TAT-HOXB4H Protein

A. Cloning and Expression

Systems for cloning and, expressing proteins in a variety of host cellsare known in the art. Cells suitable for producing proteins aredescribed in, for example, Fernandez et al. (1999) Gene ExpressionSystems, Academic Press, eds. In brief, suitable host cells includemammalian cells, insect cells, plant cells, yeast cells, or prokaryoticcells, e.g., E. coli. Mammalian cells available in the art forheterologous protein expression include lymphocytic cell lines (e.g.,NSO), HEK293 cells, Chinese hamster ovary (CHO) cells, COS cells, HeLacells, baby hamster kidney cells, oocyte cells, and cells from atransgenic animal, e.g., mammary epithelial cell. Suitable vectors maybe chosen or constructed to contain appropriate regulatory sequences,including promoter sequences, terminator sequences, polyadenylationsequences, enhancer sequences, marker genes, and other sequences. Thevectors may also contain a plasmid or viral backbone. For details, seeSambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., ColdSpring Harbor Laboratory Press (1989). Many established techniques usedwith vectors, including the manipulation, preparation, mutagenesissequencing, and transfection of DNA, are described in Current Protocolsin Molecular Biology, Second Edition, Ausubel et al. eds., John Wiley &Sons (1992).

A further aspect of the disclosure provides a method of introducing thenucleic acid into a host cell. For eukaryotic cells, suitabletransfection techniques may include calcium phosphate, DEAE-Dextran,electroporation, liposome-mediated transfection, and transduction usingretrovirus orother viruses, e.g., vaccinia or baculovirus. For bacterialcells, suitable techniques may include calcium chloride transformation,electroporation, and transfection using bacteriophage. DNA introductionmay be followed by a selection method (e.g., drug resistance) to selectcells that contain the nucleic acid.

B. Purification and Refolding

TAT-HOXB4H protein can be initially isolated from the recombinant hostcell by any appropriate means known in the art. For example, the proteincan be removed from cell supernatant, if the protein is capable of beingsecreted, or the protein can be removed from a cell lysate.

TAT-HOXB4H protein can be purified using the chromatographic methodscomprising: (a) applying the cell lysate or cell supernatant, if theprotein is being secreted into the solution, to a chromatography columnfor purifying histidine-tagged proteins; b) washing the chromatographycolumn for purifying histidine-tagged proteins with a buffer; (c)eluting the partially purified protein from the chromatography columnfor purifying histidine-tagged proteins; (d) applying the partiallypurified protein obtained from chromatography column for purifyinghistidine-tagged proteins to a cation ion exchange chromatographycolumn; (e) washing the cation ion exchange chromatography column with abuffer; (f) eluting the purified TAT-HOXB4H protein from the cation ionexchange chromatography column.

Cell lysate or cell supernatant, if the protein is capable of beingsecreted into the solution, can be cleared by centrifugation at 20,000×gfor 30 min at 4° C., the supernatant adjusted to 10 mM imidazole andloaded on the chromatography chelating columns for purifyinghistidine-tagged proteins (Amersham Pharmacia). The column can be washedwith 8 M urea,.20 mM HEPES, 0.5 mM DTT, 10 mM NaCl, pH 8.0 and 10 mMimidazole to remove unbound proteins. Partially pure TAT-HOXB4 proteincan be eluted from the chromatography column for purifyinghistidine-tagged proteins with high concentration of imidazole and salt.

For further purification, partially purified protein obtained fromchromatography column for purifying histidine-tagged proteins can beapplied to cation ion exchange chromatography column (AmershamPharmacia). The column can be washed with 4 M urea, 20 mM HEPES, 50 mMNaCl, pH 6.5 to remove unbound proteins. Bound TAT-HOXB4H can be elutedwith high salt. The purified TAT-HOXB4H protein collected from thispurification procedure is in the denatured form.

Further, the denatured TAT-HOXB4H protein eluted from the cation ionexchange chromatography column can be refolded using hydrophobiccompounds by (i) combining the eluted denatured protein and a solutionof hydrophobic compounds to form a solution of protein and hydrophobiccompounds; (ii) desalting the solution of protein and hydrophobiccompounds to obtain a desalted protein and hydrophobic compoundsolution; and (iii) removing the hydrophobic compounds from the desaltedprotein solution using ultrafiltration.

As used in the present invention, the term “hydrophobic compounds” referto any hydrophobic compounds capable of protecting the desired proteinfrom forming insoluble aggregates during the denaturing salt-removingstep. Hydrophobic compounds suitable for use in the present inventionare described in Oganesyan et al., Pharmagenomics (2004) 71, 22-26.Suitable hydrophobic compounds include, but are not limited to, TritonX-100, tween-20 or polybenzene compounds. Ultrafiltration or bufferexchange can be carried out by a centricon or stir-cell. The conditionsfor ultrafiltration or buffer exchange may vary, as recognized by thoseskilled in the art, depending on the types of the desired protein.

In one embodiment of the present invention, the hydrophobic compound inthe desalted solution containing denatured HOXB4H protein is removed by5-10 times of buffer exchange (each performed by centrifugation at1000-2500×g for 10 min) with solution containing low to highconcentrations of large hydrophobic compounds such as beta-cyclodextrinwhereby, the denatured HOXB4H protein is refolded into native formthereof.

In one embodiment, purified TAT-HOXB4H protein can be stored incommercially available IMDM (HyClone) medium (storage buffer 1) at 4° C.or -20° C.

In another embodiment, purified TAT-HOXB4H protein can be stored incommercially available DMEM (HyClone) medium (storage buffer 2) at 4° C.or -20° C.

In one embodiment, His tag at the C-terminus of TAT-HOXB4H may beremoved before in vivo administration.

In another embodiment His tag at the N-terminus of TAT-HOXB4H can beremoved before in vivo administration.

In another embodiment both His tags at the N- and C- termini can beremoved before in vivo administration.

C. Preparation of a Pharmaceutical Composition

TAT-HOXB4H may be used as a pharmaceutical composition when combinedwith a pharmaceutically acceptable carrier. Such a composition maycontain, in addition to the TAT-HOXB4H protein and carrier, variousdiluents, fillers, salts, buffers, stabilizers, solubilizers, and othermaterials well known in the art. The term “pharmaceutically acceptable”means a non-toxic material that does not interfere with theeffectiveness of the biological activity of the active ingredient(s).The characteristics of the carrier may depend on the route ofadministration.

It is especially advantageous to formulate compositions in dosage unitform for ease of administration and uniformity of dosage. Dosage unitform as used herein refers to physically discrete units.suited asunitary dosages for the subject to be treated; each unit containing apredetermined quantity of active compound calculated to produce thedesired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on the uniquecharacteristics of the active compound and the particular therapeuticeffect to be achieved, and the limitations inherent in the art ofcompounding such an active compound for the treatment of individuals.

Typical routes of administration include, without limitation, oral,topical, parenteral (e.g., sublingually or buccally), sublingual,rectal, vaginal, and intranasal. The term parenteral as used hereinincludes subcutaneous injections, intravenous, intramuscular,intrasternal, intracavernous, intrathecal, intrameatal, intraurethralinjection or infusion techniques. The pharmaceutical composition isformulated so as to allow the active ingredients contained therein to bebioavailable upon administration of the composition to a patient.Compositions that may be administered to a patient take the form of oneor more dosage units, where for example, a tablet may be a single dosageunit, and a container of one or more compounds of the invention inaerosol form may hold a plurality of dosage units.

When a therapeutically effective amount of a TAT-HOXB4H protein isadministered orally, the binding agent may be in the form of a tablet,capsule, powder, solution or elixir. When administered in tablet form,the pharmaceutical composition of the invention may additionally containa solid carrier such as a gelatin or an adjuvant. The tablet, capsule,and powder contain from about 5 to 95% binding agent, and preferablyfrom about 25 to 90% binding agent. Examples are sucrose, kaolin,glycerin, starch dextrins, sodium alginate, carboxymethylcellulose andethyl cellulose. Coloring and/or flavoring agents may be present. Acoating shell may be employed. When administered in liquid form, aliquid carrier such as water, petroleum, oils of animal or plant originsuch as peanut oil, mineral oil, soybean oil, or sesame oil, orsynthetic oils may be added. The liquid form of the pharmaceuticalcomposition may further contain physiological saline solution, dextroseor other saccharide solution, or glycols such as ethylene glycol,propylene glycol or polyethylene glycol. When administered in liquidform, the pharmaceutical composition contains from about 0.5 to 90% byweight of the binding agent, and preferably from about 1 to 50% thebinding agent.

When a therapeutically effective amount of TAT-HOXB4H protein isadministered by intravenous, cutaneous or subcutaneous injection,binding agent may be in the form of a pyrogen-free, parenterallyacceptable aqoeous solution. The preparation of such parenterallyacceptable protein solutions, having due regard to pH, isotonicity,stability, and the like, is within the skill in the art. In someembodiments, pharmaceutical composition for intravenous, cutaneous, orsubcutaneous injection may contain, in addition to binding agent anisotonic vehicle such as Sodium Chloride Injection, Ringer's Injection,Dextrose Injection, Dextrose and Sodium Chloride Injection, LactatedRinger's Injection, or other vehicle as known in the art. Thepharmaceutical composition of the present invention may also containstabilizers, preservatives, buffers, antioxidants, or other additiveknown to those of skill in the art.

In practicing the method of treatment or use of the present invention, atherapeutically effective amount of TAT-HOXB4H protein is administeredto a subject, e.g., mammal (e.g., a human). As used herein, the term“therapeutically effective amount” means the total amount of each activecomponent of the pharmaceutical composition or method that is sufficientto show a meaningful patient benefit, e.g., amelioration of symptoms of,healing of, or increase in rate of healing of such conditions. Whenapplied to an individual active ingredient, administered alone, the termrefers to that ingredient alone. When applied to a combination, the termrefers to combined amounts of the active ingredients that result in thetherapeutic effect, whether administered in combination, serially orsimultaneously.

The amount of TAT-HOXB4H protein in the pharmaceutical composition ofthe present invention may depend upon the nature and severity of thecondition being treated, and on the nature of prior treatments that thepatient has undergone, and patient's age and sex. Ultimately, theattending physician may decide the amount of active ingredient withwhich to treat each individual patient. Initially, the attendingphysician may administer low doses of active ingredient and observe thepatient's response. Larger doses of active ingredient may beadministered until the optimal therapeutic effect is obtained for thepatient, and at that point the dosage is not generally increasedfurther. It is contemplated that the various pharmaceutical compositionsused to practice the method of the present invention may contain about 1μg to about 1 mg TAT-HOXB4H protein per kg body weight. Examples ofdosage ranges that can be administered to a subject can be chosen from:1 μg/kg to 1 mg/kg, 1 μg/kg to 0.5 mg/kg, 1 μg/kg to 0.1 mg/kg, 10 μg/kgto 0.5 mg/kg, 10 μg/kg to 0.1 mg/kg, 100 μg to 0.5 mg/kg, 250 μg/kg to0.5 mg/kg. Further, examples of dosage ranges that can be chosen from:50 μg to 100 mg, 100 μg to 50 mg, 500 μg to 50 mg, 1 mg to 50 mg. Theduration of intravenous therapy using the pharmaceutical composition ofthe present inention may vary, depending on the severity of the diseasebeing treated and the condition and potential idiosyncratic response ofeach individual patient. In one embodiment, it is contemplated that theduration of each application of the TAT-HOXB4H protein may be in therange of 12 to 24 hours of continuous intravenous administration. Inanother embodiment, the duration of application of TAT-HOXB4H proteinmay last as long as patient's radiation or chemotherapy continues.TAT-HOXB4 protein may be administered in the range of 10-100 μg/kgintravenously, twice a day for 4.5 to 5 days. One cycle of the treatmentmay be enough to expand HSCs in vivo. Ultimately the attending physicianmay decide on the appropriate duration of intravenous therapy using thepharmaceutical composition of the present invention.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Data obtained from the cell culture assays and animal studies can beused in evaluating a range of dosage for use in humans. The dosage ofsuch compounds may lie within a range of circulating concentrations thatinclude the ED₅₀ with little or no toxicity. The dosage may vary withinthis range depending upon the dosage form employed and the route ofadministration utilized. The therapeutically effective dose ofTAT-HOXB4H can be estimated initially from cell culture assays. A dosemay be formulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test protein which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Levels-in plasma may be measured, forexample, by high performance liquid chromatography. The effects of anyparticular dosage can be monitored by a suitable bioassay. Examples ofsuitable bioassays include, but not limited to, measuring CD34⁺ stemcells in the mononuclear cell by using fluorescent probe (e.g., FITC)tagged antibodies to CD34+ stem cells and measuring the percentage ofcells in the peripheral blood or bone marrow HSCs by flow cytometry. Thepolynucleotide and proteins of the present invention are expected toexhibit one or more of the uses or biological activities (includingthose associated with assays cited herein) identified below. Uses oractivities described for proteins of the present invention may beprovided by administration or use of such proteins or by administrationor use of polynucleotides encoding such proteins (such as, for example,in gene therapies or vectors suitable for introduction of DNA).

III. Methods of Stimulating Hematopoiesis In Vivo

A. Patients in Need of Treatment

The pharmaceutical compositions of the invention may be used to treatdiseases which include, but are not limited to, autoimmune disorders,immunodeficiency disorders, and hematological disorders. Additionally,pharmaceutical compositions of the invention may be used to improve therecovery time after HSC transplantation.

The pharmaceutical composition of the invention include, but are notlimited to, the treatment of patient suffering from or susceptible tolymphomas, leukemias, Hodgkin's disease and myeloproliferativedisorders. Additionally, inherited diseases caused by HSC deficiency andaplastic anemia may be treated by the pharmaceutical composition of thepresent invention.

Further the pharmaceutical composition of the present invention may beemployed to the HSC donors and G-CSF-insensitive patients.

In one embodiment of the invention, TAT-HOXB4H is the only active agentadministered for mobilization of HSCs, and fluorouracil (5-FU) is notadministered to the donor, either as pretreatment or a combinationtherapy scheme.

Additional diseases or conditions associated with increased cellsurvival, that may be treated by the pharmaceutical composition of theinvention include, but are not limited to, progression, and/ormetastases of malignancies and related disorders such as leukemia(including acute leukemias (for example, acute lymphocytic leukemia,acute myelocytic leukemia (including myeloblastic, prmyelocytic,myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias(for example, chronic myelocytic (granulocytic) leukemia and chroniclymphocytic leukemia)), myelodysplastic syndrome polycythemia vera,lymphomas (for example, Hodgkin's disease and non-Hodgkin's disease),multiple myeloma, Waldenstrom's macroglobulinemia, and solid tumorsincluding, but not limited to, sarcomas and carcinomas such asfibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma,.choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, andretinoblastoma.

The present invention is not limited to the particular methodology,protocols, cell lines, animal species or genera, and reagents describedbelow. The terminology used to describe particular embodiments is notintended to limit the scope of the present invention, which may belimited only by the appended claims. As used herein, the singular forms“a,” “and,” and “the” include plural reference unless the contextclearly dictates otherwise. Thus, for example, reference to “a cell” isa reference to one or more cells and includes equivalents thereof knownto those skilled in the art.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention belongs. Although any methods, devices,and materials similar or equivalent to those described herein can beused in the practice or testing of the invention, the preferred methods,devices, and materials are now described. All publications and patentsmentioned herein are hereby ,incorporated herein by reference for thepurpose of describing and disclosing, for example, the constructs andmethodologies that are described in the publications which might be usedin connection with the presently described invention. The publicationsdiscussed above and throughout the text are provided solely for theirdisclosure prior to the effective date of the present application.Nothing herein is to be construed as an admission that the inventors arenot entitled to antedate such disclosure by virtue of prior invention.

Definitions

A “stem cell” is a pluripotent or multipotent cell with the ability toself-renew, to remain undifferentiated, and to become differentiated.Stem cells can divide without limit, at least for the lifetime of theanimal in which they naturally reside. Stem cells are not terminallydifferentiated, i.e., they are not at the end of a pathway ofdifferentiation. When a stem cell divides, each daughter cell can eitherremain a stem cell or it can embark on a course that leads to terminaldifferentiation. A “chimeric” stem cell is a stem cell with a portion ofits DNA belonging to a heterologous organism.

A “hematopoeitic” cell is a cell involved in the process ofhematopoeisis, i.e., the process of forming mature blood cells fromprecursor cells. In the adult, hematopoeisis takes place in the bonemarrow. Earlier in development, hematopoeisis takes place at differentsites during different stages of development; primitive blood cellsarise in the yolk sac, and later, blood cells are formed in the liver,spleen, and bone marrow. Hematopoeisis undergoes complex regulation,including regulation by hormones, e.g., erythropoietin; growth factors,e.g., colony stimulating factors; and cytokines, e.g., interleukins.

The term “vector”, as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been-linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply, “expression vectors”). In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” may be used interchangeably as the plasmid is the most commonlyused form of vector.

However, the invention is intended to include such other forms ofexpression vectors, such as viral vectors (e.g., replication defectiveretroviruses, adenoviruses and adeno-associated viruses), which serveequivalent functions.

“Transformation,” as used herein, refers to the insertion of anexogenous polynucleotide into a host cell, irrespective of the methodused for insertion: for example, transformation by direct uptake,transfection, infection, and the:like. For particular methods oftransfection, see further below. The exogenous polynucleotide may bemaintained as a nonintegrated vector, for example, an episome, oralternatively, may be integrated into the host genome.

In general, the term “protein” refers to any polymer of two or moreindividual amino acids (whether or not naturally occurring) linked viapeptide bonds, as occur when the carboxyl carbon atom of the carboxylicacid group bonded to the α-carbon of one amino acid (or amino acidresidue) becomes covalently bound to the amino nitrogen atom of theamino group bonded to the α-carbon of an adjacent amino acid. Thesepeptide bond linkages, and the atoms comprising them (i.e., α-carbonatoms, carboxyl carbon atoms (and their substituent oxygen atoms), andamino nitrogen atoms (and their substituent hydrogen atoms)) form the“polypeptide backbone” of the protein. In addition, as used herein, theterm “protein” is understood to include the terms “polypeptide” and“peptide” (which, at times, may be used interchangeably herein).Similarly, protein fragments, analogs, derivatives, and variants are maybe referred to herein as “proteins,” and shall be deemed to be a“protein” unless otherwise indicated. The term “fragment” of a proteinrefers to a polypeptide comprising fewer than all of the amino acidresidues of the protein. As may be appreciated, a “fragment” of aprotein may be a form of the protein truncated at the amino terminus,the carboxy terminus, and/or internally (such as by natural splicing),and may also be variant and/or derivative. A “domain” of a protein isalso a fragment, and comprises the amino acid residues of the proteinrequired to confer biochemical activity corresponding to naturallyoccurring protein.

“Recombinant” as used herein to describe a nucleic acid molecule means apolynucleotide of genomic, cDNA, viral, semisynthetic, or syntheticorigin which, by virtue of its origin or manipulation is not associatedwith all or a portion of the polynucleotide with which it is associatedin nature. The term “recombinant” as used with respect to a protein orpolypeptide means a polypeptide produced by expression of a recombinantpolynucleotide.

An “isolated,” “purified,” “substantially isolated,” or “substantiallypure” molecule (such as a polypeptide or polynucleotide) is one that hasbeen manipulated to exist in a higher concentration than in nature. Forexample, a subject protein is isolated, purified, substantiallyisolated, or substantially purified when at least 50%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more ofnon-subject-protein materials with which it is associated in nature havebeen removed. As used herein, an “isolated,” “purified,” “substantiallyisolated,” or “substantially purified” molecule includes recombinantmolecules.

A “SCID mouse” is a mouse model for severe combined immunodeficiencysyndrome (SCID), which causes severe defects in the development of theimmune system. These mice are deficient in, or completely lack; both Tand B lymphocytes. The SCID mutation appears to impair the recombinationof antigen receptor genes, causing a lack of functional T and Blymphocytes. Other hematopoietic cell types appear to develop andfunction normally. SCID mice readily support normal lymphocytedifferentiation and can be reconstituted with normal lymphocytes fromsyngeneic or allogeneic Mice, or with human lymphocytes. These mice alsosupport the growth of allogeneic and xenogeneic tumors. Therefore, SCIDmice, which allow disseminated growth of a number of human tumors,particularly hematologic disorders and malignant melanoma, can be usedto investigate malignancies.

The terms “subject,” “individual,” “host,” and “patient” are usedinterchangeably herein to refer to a living animal, including a humanand a non-human animal. The subject may, for example, be an organismpossessing immune cells capable of responding to antigenic stimulation,and stimulatory and inhibitory signal transduction through cell surfacereceptor binding. The subject may be a mammal, such as a human ornon-human mammal, for example, dogs, cats, pigs, cows, sheep, goats,horses, rats, and mice. The term “subject” does not preclude individualsthat are entirely normal with respect to a disease, or normal in allrespects.

The term “treatment” refers to a therapeutic or preventative measure.The treatment may be administered to a subject having a medical disorderor who ultimately may acquire the disorder, in order to prevent, cure,delay, reduce the severity of, or ameliorate one or more symptoms of adisorder or recurring disorder, or in order to prolong the survival of asubject beyond that expected in the absence of such treatment.

The term “therapeutically effective amount” means the amount of thesubject compound that may elicit a desired response, for example, abiological or medical response of a tissue, system, animal, or humanthat is sought, for example, by a researcher, veterinarian, medicaldoctor, or other clinician.

EXAMPLES

The following specific examples are to be construed as merelyillustrative, and not imitative of the remainder of the disclosure inany way whatsoever. Without further elaboration, it is believed that oneskilled in the art can, based on the description herein, utilize thepresent invention to its fullest extent.

Example 1 Construction of Plasmid pET21b-His-pTAT-HOXB4-His

(a) The Modification of pET21b Plasmid Containing N- and C-Terminal HisTag and TAT Signal Peptide.

The expression vector pET21 b containing N-terminal His tag and a TATsignal peptide was generated by inserting oligonucleotides5’-TATGCACCACCACCACCACCACTACGGCCGCAAGAAACGCCGCCAGCGC CGCCGGCG-3′ (sense)(SEQ ID NO: 1) and 5′-CTAGCGGCGCTGGCGGCGTTTCTTGCGGCCGTAGTGGTGGTGGTGGTGGCCA-3′ (antisense) (SEQ ID NO: 2) into pET21b plasmid. The C-terminalHis-Tag is already present in the pET21b vector.

(b) Cloning of HOXB4 into Modified pET21 b Expression Vector

A DNA fragment containing the open reading frame (ORF) of HOXB4 andadditional 6 histidine (SEQ ID NO: 5) coding sequence was obtained byPCR amplification using plasmid MGC54130 (GeneDiscovery, Taipei, Taiwan.Cat. No. 5533346) as the template, and the PCR-generated HOXB4 cDNAfragment was subcloned into the modified pET21 b expression vector.Plasmid construction and nucleic acid sequences are shown in FIGS. 2 and3.

Example 2 Expression of Recombinant TAT-HOXB4H Protein in E. coli

The pET21b-His-TAT-HOXB4-His expression vector was transformed into E.coli strain BL21(DE3)pLysS (Novagen). The transformed cells were grownovernight at 37° C. The overnight-grown cultures were diluted to aninitial OD600 of 0.05. The cultures were then grown to an OD600 of 0.5at 37° C., induced with 1 mM isopropyl-beta-D-thiogalactopyranoside(IPTG) at 37° C. for 3 hr, with vigorous shaking.

Example 3 Purification of Recombinant TAT-HOXB4H Protein

Following induction, cells were harvested by centrifugation andresuspended in Buffer A (8 M Urea, 20 mM HEPES, 0.5 mM DTT and 100 mMNaCl, pH 8.0). The cell suspension was passed three times through aFrench press, and the cell lysate cleared by centrifugation at 20,000 xg for 30 min at 4° C., adjusted the supernatant to 10 mM imidazole andloaded on the chromatography chelating columns for purifyinghistidine-tagged proteins (Amersham Pharmacia). Bound proteins wereeluted with 50, 100 and 250 mM imidazole in buffer A. TAT-HOXB4Hcontaining fractions were loaded on a cation ion exchange chromatographycolumn in Buffer B (4 M urea, 20 mM HEPES and 50 mM NaCl, pH 6:5),eluted with 1.5 M NaCl and 20 mM HEPES (pH 8.0).

Example 4 Renaturation of Recombinant TAT-HOXB4H Protein

The TAT-HOXB4H protein in the eluted fractions was solubilized anddenatured in a solution containing denaturing salt (e.g., guanidinehydrochloride) and then it was mixed with D-PBS-T buffer (0.1% TritonX-100 in 2×PBS). The ratio of the TAT-HOXB4H protein solution to D-PBS-Tbuffer was 1:4. The resultant mixture was added to a 10Kcentrifugalfilter tube (50 ml or 15 ml) pretreated with water (10 ml or 3 ml), andthen centrifuged at 3000 rpm for 10 min. In this step, the denaturingsalt is replaced by D-PBS-T buffer in which Triton X-100 is capable ofbinding with the hydrophobic region of the HOXB4H protein.

This step of ultrafiltration or buffer exchange using 10K centrifugalfilter was performed ten times with solution containing differentconcentrations of beta-cyclodextrin (two times of each 1 mM, 2 mM, 3 mM,4 mM and 5 mM beta-cyclodextrin in storage buffer IMDM by centrifugingat 1000-2500×g for 10 min. The remaining sample in the 10K centrifugalfilter tube was collected and stored at -80° C.

Homogeneity of the purified TAT-HOXB4H protein was analyzed bySDS-polyacrylamide gel followed by coomassie staining (FIG. 5). As shownin FIG. 5, TAT-HOXB4H purified from chromatography for'purifyinghistidine-tagged proteins and cation ion exchange chromatographyresulted in.3-4 fold yield compared to that of the TAT-HOXB4 protein.The pTAT-HA-HOXB4 plasmid was a gift from Dr. Guy Sauvageau, Universityof Montreal, Canada. This plasrnid was transformed into BL21(DE3)pLysS(Novagen) and the purification of TAT-HOXB4. protein was performed asdescribed in Krosl et al, 2003.

Example 5 Stability of Recombinant TAT-HOXB4H Protein

The Stability of the TAT-HOXB4H was measured by SDS-polyacrylamide gelanalysis. Upon storage full length TAT-HOXB4H can be degraded into 30 kDand 10 kD fragments. As shown in FIG. 6, TAT-HOXB4H protein produced bythe method of this invention was stable even after 16 hours stored inPBS at 4° C.

Further, the stability of TAT-HOXB4H protein stored at 4° C. and −20° C.in PBS and storage buffer IMDM was analyzed by 10% SDS-polyacrylamidegel electrophoresis followed by coomassie staining. As shown in FIG. 7,when IMDM was used as a storage buffer, the stability of the TAT-HOXB4Hprotein was maintained even after 4 weeks.

Example 6 Effect of Recombinant TAT-HOXB4H Protein on Hematopoiesis inWild Type Balb/c Mice

Balb/c mice were used to investigate the possible effect of recombinantTAT-HOXB4H protein on mobilization of HSCs from bone marrow toperipheral blood. TAT-HOXB4H recombinant protein in phosphate bufferedsaline (PBS) was administered by subcutaneous injection four times perday for 4 days. To examine dose-responsiveness, experimental groups(n=21) received a dose ranged from 1 μg, 5 μg, 10 μg, 15 μg . . . to 100μg per kg BW per mouse. A separate group of control mice receivedphosphate buffered saline only, and another group of control mice wereinjected subcutaneously twice per day for 4 day with a dose of 5 μg perkg BW of G-CSF per mouse.

Peripheral blood was harvested from all mice and analyzed by a flowcytometer to obtain the percentage of CD34⁺ stem cells in mononuclearcell (MNC). The results are reported as the mean±s.d. in Table 1.

TABLE 1 Group (Experimental/Control) TAT-HOXB4H (μg/kg) CD34⁺/MNC (%)  11   0 ± 0.03  2 5  0.3 ± 0.05  3 10 0.45 ± 0.03  4 15 0.42 ± 0.01  5 200.38 ± 0.05  6 25 0.41 ± 0.02  7 30 0.35 ± 0.21  8 35 0.33 ± 0.11  9 400.29 ± 0.16 10 45 0.46 ± 0.01 11 50 0.45 ± 0.02 12 55 0.42 ± 0.06 13 600.44 ± 0.02 14 65 0.41 ± 0.04 15 70 0.41 ± 0.03 16 75 0.49 ± 0.01 17 800.45 ± 0.04 18 85 0.46 ± 0.07 19 90 0.44 ± 0.02 20 95 0.41 ± 0.01 21 1000.42 ± 0.05 Control (PBS) 0 0.002 Control (G-CSF) 0  0.5 ± 0.03

The percentage of CD34⁺/MNC in peripheral blood harvested from thetreated mice is shown in Table 1. The TAT-HOXB4H treated experimentalgroups 3-21 (received a dose of 10 μg or above per kg BW) showedsubstantially the same mobilization effect as G-CSF treated controlgroup.

Bone marrow from TAT-HOXB4H treated mice (Exp. Group 3 received a doseof 10 μg per kg BW), G-CSF treated mice, and PBS injected mice werefurther phenotyped using FITC-conjugated antibody to CD34⁺ (BectonDickinson) and analyzed by a flow cytometer. Bone marrow from micetreated with TAT-HOXB4H (FIG. 5C) appears to be richer in CD34⁺ stemcells than bone marrow from G-CSF (FIG. 5A) and PBS (FIG. 5B) injectedmice. Therefore, these results indicate that injection of recombinantTAT-HOXB4H proteins results in increased number of HSCs in both the bonemarrow and peripheral blood in mice.

Example 7 Effect of Recombinant TAT-HOXB4H Protein on Hematopoiesis inRhesus Monkey

Male adult rhesus monkeys were used to investigate the efficacy ofrecombinant TAT-HOXB4H protein in monkeys. Experimental group I (n=5)were injected intravenously with 10 μg per kg BW of recombinantTAT-HOXB4H protein four times per day for 4 days. Experimental group II(n=5) were injected intravenously with 10 μg per kg BW of TAT-HOXB4Hfour times per day and injected subcutaneously with 5 μg per kg BW ofG-CSF for 4 days. Control group I received PBS only, and control groupII were injected subcutaneously twice per day for 4 day with a dose of 5μg per kg BW of G-CSF. Peripheral blood were harvested from all monkeysand analyzed by a flow cytometer to obtain the percentage of CD34⁺ stemcells in mononuclear cell (MNC). The results are presented in Table 2.

TABLE 2 Group (Experimental/Control) CD34⁺/MNC (%) I. TAT-HOXB4H (10μg/kg) 0.62 II. TAT-HOXB4H (10 μg/kg) + G-CSF (5 μg/kg) 0.38 Control 1(PBS) 0.07 Control 2 (G-CSF, 5 μg/kg) 0.28

As shown in Table 2, monkeys (experimental group I) treated withTAT-HOXB4H only showed a significantly better mobilization effect thanG-CSF treated monkeys (control group II). The monkeys treated withTAT-HOXB4H and G-CSF (experimental group II) showed a slightly bettermobilization effect than G-CSF treated monkeys (control group II).

Bone marrow specimens from monkeys treated with TAT-HOXB4H, G-CSF or PBSwere further phenotyped using FITC-conjugated antibody to CD34⁺ (BectonDickinson) and analyzed by a flow cytometer. Bone marrow specimens frommonkeys treated with TAT-HOXB4H (FIG. 6C) appears significantly richerin CD34⁺ stem cells than bone marrow specimens from G-CSF (FIG. 6A),TAT-HOXB4H 30 G-CSF (FIG. 6B) and PBS (FIG. 6D) injected monkeys.

Example 8 Effect of Recombinant TAT-HOXB4H Protein on HematopoieticRecovery in NOD-SCID Mice

10⁴ human Lin⁻/CD34⁺ cells were injected into irradiated (2.5 Gy)NOD-LtSz-scid/scid (NOD-SCID) mice, along with 10⁵ CD34⁻ irradiatedaccessory cells. The mice are divided into two groups randomly: onegroup (n=28) was injected intravenously with 10 μg per kg BW ofrecombinant TAT-HOXB4H protein twice per day, and the other (n=28)received PBS twice per day. The presence of human CD45⁺ cells in theperipheral blood cells of all mice was measured periodically by flowcytometry after transplantation. Hematopoietic recovery was evaluated asthe number of mice whose human CD45⁺ cells reached levels of >0.1% inperipheral blood (PB) after transplantation. As shown in FIG. 10,improved hematopoietic recovery was observed in the mice injected withrecombinant TAT-HOXB4H protein.

Example 9 Effect of Recombinant TAT-HOXB4H Protein on HematopoieticRecovery in Balb/c mice After Cisplatin Chemotherapy

5 week-old Balb/c mice were repeatedly injected intravenously withcisplatin until the number of Ly5 (murine CD45) cells in peripheralblood of the mice decreased to approximately 10% of the original number.The mice treated with cisplatin are divided into two groups randomly:one group (n=28) was injected intravenously with 10 μg per kg BW ofrecombinant TAT-HOXB4H protein twice per day, and the other (n=28)received PBS twice per day. The presence of Ly5 cells in the peripheralblood cells of all mice was measured periodically by flow cytometryafter transplantation. Hematopoietic recovery rate was evaluated as thepercentage of the number of Ly5 cells in peripheral blood to theoriginal number. As shown in FIG. 8, improved hematopoietic recovery wasobserved in the mice injected with recombinant TAT-HOXB4H protein.

The animal models used in these experiments have been recognized in theart as predictive of results that will be obtained in human patients.See, e.g., Broxmeyer et al. (2005) The Journal of Experimental Medicine,201, 1307-1318; Larochelle et al. (2006) Blood 107, 3772-3778.

Although the invention has been explained in relation to its variousembodiments, it is to be understood that many other possiblemodifications and variations can be made without departing from thespirit and scope of the invention as hereinafter claimed.

1-10. (canceled)
 11. A TAT-HOXB4H protein having the amino acid sequenceof SEQ ID NO: 4 produced by a method comprising: (a) providing anisolated host cell comprising a vector encoding the protein; (b)expressing the protein in the host cell; (c) collecting an impuresolution of the expressed protein; (d) purifying the protein from theimpure solution by: (1) applying the impure solution to a chromatographycolumn for purifying histidine-tagged proteins; (2) washing thechromatography column for purifying histindine-, tagged proteins with awashing buffer; (3) eluting the protein from the chromatography columnfor purifying histidine-tagged proteins with an eluting, buffer to forma partially purified protein solution; (4) applying the partiallypurified protein solution to a cation ion exchange chromatographycolumn; (5) washing the cation ion exchange chromatography column with awashing buffer; and (6) eluting the purified protein from the cation ionexchange chromatography column in denatured form with an eluting buffer;(e) refolding the eluted denatured protein using hydrophobic compoundsby: (1) combining the eluted denatured protein and a solution of:hydrophobic compounds to form a solution of the protein and hydrophobiccompounds; (2) desalting the solution of the protein and hydrophobiccompounds to obtain a desalted protein and hydrophobic compoundsolution; (3) removing the hydrophobic compounds from the desaltedprotein and hydrophobic compound solution using ultrafiltration by acentrifugal filter tube or a stir-cell; and (f) storing the purifiedprotein in Iscove's modified Dulbecc's medium (IMDM) or Dulbecco'smodified eagle medium (DMEM). 12-27. (canceled)
 28. The method of claim11, wherein the chromatography column for purifying histidine-taggedproteins washing buffer comprises 8 M Urea, 20 mM HEPES, 0.5 mM DTT, 100mM NaCl, pH 8.0 and 10 mM imidazole.
 29. The method of claim 11, whereinthe eluting buffer to elute the protein from the chromatography columnfor purifying histidine-tagged proteins to form a partially purifiedprotein solution comprises 50 mM imidazole in 8 M Urea, 20 mM HEPES, 0.5mM DTT, 100 mM NaCl, pH 8.0.
 30. The method of claim 11, wherein theeluting buffer to elute the protein from the chromatography column forpurifying histidine-tagged proteins to form a partially purified proteinsolution comprises 50-100 mM imidazole.
 31. The method of claim 11,wherein the eluting buffer to elute the protein from the chromatographycolumn for purifying histidine-tagged proteins to form a partiallypurified protein solution comprises 100-250 mM imidazole.
 32. The methodof claim 11, wherein the cation ion exchange chromatography columnwashing buffer comprises 4 M urea, 20 mM HEPES and 50 mM NaCl, pH 6.5.33. The method of claim 11, wherein the eluting buffer to elute thepurified protein from the cation ion exchange chromatography columncomprises 1.5 M NaCl and 20 mM HEPES, pH 8.0.
 34. The method of claim11, wherein the hydrophobic compounds comprise Triton X-100, tween-20 orpolybenzene compounds.
 35. The method of claim 11, wherein thecentrifugal filter tube is a 10K centrifugal filter tube.
 36. The methodof claim 11, wherein the ultrafiltration process is performed withsolutions containing different concentrations of beta-cyclodextrin. 37.A TAT-HOXB4H protein having the amino acid sequence of positions 8 to273 of SEQ ID NO: 4 produced by a method comprising: (a) providing anisolated host cell comprising a vector encoding the protein with ahistidine tag (His-tag) at the N-terminus, the C-terminus, or both theN- and C-terminus of the protein; (b) expressing the protein in the hostcell; (c) collecting an impure solution of the expressed protein; (d)purifying the protein from the impure solution by: (1) applying theimpure solution to a chromatography column for purifyinghistidine-tagged proteins; (2) washing the chromatography column forpurifying histindine-tagged proteins with a washing buffer; (3) elutingthe protein from the chromatography column for purifyinghistidine-tagged proteins with an eluting buffer to form a partiallypurified protein solution; (4) applying the partially purified proteinsolution to a cation ion exchange chromatography column; (5) washing thecation ion exchange chromatography column with a washing buffer; and (6)eluting the purified protein from the cation ion exchange chromatographycolumn in denatured form with an eluting buffer; (e) refolding theeluted denatured protein using hydrophobic compounds by: (1) combiningthe eluted denatured protein and a solution of hydrophobic compounds toform a solution of the protein and hydrophobic compounds; (2) desaltingthe solution of the protein and hydrophobic compounds to obtain adesalted protein and hydrophobic compound solution; (3) removing thehydrophobic compounds from the desalted protein and hydrophobic compoundsolution using ultrafiltration by a centrifugal filter tube or astir-cell; (f) removing a His-tag from at least one end of the protein;and (g) storing the purified protein in Iscove's modified Dulbecco'smedium (IMDM) or Dulbecco's modified eagle medium (DMEM).
 38. The methodof claim 37, wherein the His-tag is removed from the N-terminus of theprotein.
 39. The method of claim 37, wherein the His-tag is removed fromthe C-terminus of the protein.
 40. A TAT-HOXB4H protein having the aminoacid sequence of SEQ ID NO:
 4. 41. A TAT-HOXB4H protein having the aminoacid sequence of positions 8 to 273 of SEQ ID NO: 4.